Polymer-Rich Dense Phase Can Concentrate Metastable Silica Precursors and Regulate Their Mineralization

Multistep mineralization processes are pivotal in the fabrication of functional materials and are often characterized by far from equilibrium conditions and high supersaturation. Interestingly, such ‘nonclassical’ mineralization pathways are widespread in biological systems, even though concentrating molecules well beyond their saturation level is incompatible with cellular homeostasis. Here, we show how polymer phase separation can facilitate bioinspired silica formation by passively concentrating the inorganic building blocks within the polymer dense phase. The high affinity of the dense phase to mobile silica precursors generates a diffusive flux against the concentration gradient, similar to dynamic equilibrium, and the resulting high supersaturation leads to precipitation of insoluble silica. Manipulating the chemistry of the dense phase allows to control the delicate interplay between polymer chemistry and silica precipitation. These results connect two phase transition phenomena, mineralization and coacervation, and offer a framework to achieve better control of mineral formation.


Reagents
Poly(acrylamide-co-acrylic acid) (Mw ~520,000) and polyethylenimine (Mw ~750,000) were purchased from Sigma-Aldrich and used without any pretreatments. All reported polymer concentrations were used as the concentration of functional groups, which were calculated using the reported polymer purities and average sizes. Sodium silicate solution ((NaOH) x (Na2SiO3) y • z H2O, 27% SiO 2 ) from Sigma-Aldrich was used as the silicon source.
Ultra-pure water (Milli-Q IQ 7003 Ultrapure Lab Water System, Merck) was used for solution preparation.
Polymer phase separation and silicification 0.25 mL PEI stock solution (200 mM, pH 5.0) was mixed with 0.25 mL PAMcoAA stock solution (200 mM, pH 5.0) and 0.5 mL milli-Q water to reach 1 mL. After 30 min the mixed solution was centrifuged at 10000 g for 3 min. Dilute phases were removed by pipette and the polymer dense phases were moved into plastic dishes for further silicification. To silicify the coacervates, 5 mL Si(OH) 4 and 10 mM PEI solution was added into the dish as a new dilute phase. The dilute phase were refreshed every 12 h.

Dynamic light scattering
Zetasizer Nano ZSP (Malvern Instruments, United Kingdom) equipped with a 633 nm laser was used for dynamic light scattering (DLS) experiments to measure particle sizes in realtime. The particle sizes were determined by intensity distribution and presented as the average values of three replicate measurements.

S3
Condensates were dissolved in 5 mL 1 M NaOH. After 24 h incubation, 100 μL were further diluted to 5 mL using milli-Q water and the silica concentration was subsequently determined using the silicate test kit (Merck Millipore, USA). The amount of silica was calculated by the measured Si-concentration × 5 mL and presented as the average values of three replicate measurements.

Thermogravimetric Analysis
Lyophilized samples were analyzed by thermal gravimetric analysis (SDT Q600, TA Instruments, USA). Analyses were performed under air atmosphere (injection rate of 100 mL/min) with a heating rate of 10 K/min. When temperature approached 800 ℃, all organic parts were combusted and dry Si-contents were calculated. The dry Si-contents present in this work were present as the average values of three replicate measurements.

SEM observations
Lyophilized samples were mounted onto SEM holders. The samples were coated with 5 nm iridium (Compact Coating Unit, CCU-010, Safematic), and imaged by SEM (Sigma, Zeiss) under 5 kV. Elemental analyzes were performed by Energy-dispersive x-ray spectroscopy (EDS) using an acceleration voltage of 5 kV and the signal from the sample was recorded using a Bruker Quantax microanalysis system equipped with an XFlash6 60mm detector.

Real-time in situ Raman monitoring
Confocal Raman Microscope (LabRam HR Evolution, Horiba) was performed to monitor Si-species in solutions and coacervates. To observe the samples, we used the ×50 objective (Olympus, LMPlanTL N) and 100 μm confocal hole. The spectra were obtained by using a laser at 532 nm as the excitation source with calibration by the characteristic band of silicon at 520.7 cm −1 and 600 lines/mm were set up to simultaneously scan a range of frequencies. The collected Raman data were analyzed by LabSpec 6 software.

Statistical analysis
The values of direct measurements are presented as average standard deviation ± collected from three independent repeats.